Siner et al. Malar J (2017) 16:417 DOI 10.1186/s12936-017-2064-9 Journal

RESEARCH Open Access Absence of inui and , but detection of and infections in asymptomatic humans in the Betong division of , Malaysian Borneo Angela Siner* , Sze‑Tze Liew, Khamisah Abdul Kadir, Dayang Shuaisah Awang Mohamad, Felicia Kavita Thomas, Mohammad Zulkarnaen and Balbir Singh*

Abstract Background: Plasmodium knowlesi, a simian malaria parasite, has become the main cause of malaria in Sarawak, Malaysian Borneo. Epidemiological data on malaria for Sarawak has been derived solely from hospitalized patients, and more accurate epidemiological data on malaria is necessary. Therefore, a longitudinal study of communities afected by knowlesi malaria was undertaken. Methods: A total of 3002 blood samples on flter paper were collected from 555 inhabitants of 8 with recently reported knowlesi malaria cases in the Betong Division of Sarawak, Malaysian Borneo. Each was visited bimonthly for a total of 10 times during a 21-month study period (Jan 2014–Oct 2015). DNA extracted from blood spots were examined by a nested PCR assay for Plasmodium and positive samples were then examined by nested PCR assays for , Plasmodium vivax, , , Plasmodium knowlesi, Plasmodium cynomolgi and . Blood flms of samples positive by PCR were also examined by microscopy. Results: Genus-specifc PCR assay detected Plasmodium DNA in 9 out of 3002 samples. Species-specifc PCR identi‑ fed 7 P. knowlesi and one P. vivax. Malaria parasites were observed in 5 thick blood flms of the PCR positive samples. No parasites were observed in blood flms from one knowlesi-, one vivax- and the genus-positive samples. Only one of 7 P. knowlesi-infected individual was febrile and had sought medical treatment at Betong Hospital the day after sam‑ pling. The 6 knowlesi-, one vivax- and one Plasmodium-infected individuals were afebrile and did not seek any medical treatment. Conclusions: Asymptomatic human P. knowlesi and P. vivax malaria infections, but not P. cynomolgi and P. inui infec‑ tions, are occurring within communities afected with malaria. Keywords: Plasmodium knowlesi, Malaria, Asymptomatic, Submicroscopic

Background Plasmodium vivax, Plasmodium ovale and Plasmodium Prior to 2004, human were thought to be caused malariae. Discovery of a large number of Plasmodium by four species of Plasmodium; Plasmodium falciparum, knowlesi cases in the division of Sarawak [1] led to the inclusion of P. knowlesi, a simian malaria parasite [2],

*Correspondence: [email protected]; [email protected] as the ffth cause of malaria in humans [3]. Morphologi- Malaria Research Centre, Faculty of Medicine and Health Sciences, cal similarities between P. knowlesi and P. malariae, when Universiti Sarawak, 94300 , Sarawak, Malaysia examined by microscopy, had led to P. knowlesi being

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Siner et al. Malar J (2017) 16:417 Page 2 of 11

misdiagnosed mainly as P. malariae [4]. Plasmodium PCR resulted in the detection of more P. knowlesi posi- knowlesi infections have been reported in other states in tive samples: 67/372 (18%), 71/335 (21.9%) and 60/289 Malaysia [5–9] and in other Southeast Asian countries, (20.7%), respectively. Only one of the 1147 asymptomatic including Tailand [10, 11], Philippines [12], Singapore individuals was positive by microscopy [29]. [13], Myanmar [14], Indonesia [15], Cambodia [16] and Plasmodium knowlesi is typically found in nature in Vietnam [17]. long-tailed and pig-tailed macaques [23]. Other simian Plasmodium knowlesi is the most common cause of malaria parasites, such as Plasmodium cynomolgi and malaria in Sarawak. Epidemiology data for 2014–2015 Plasmodium inui that infect these macaques, have also showed that P. knowlesi was responsible for 82.9% of the been shown to be capable of inducing malaria in humans 2174 malaria cases diagnosed in Sarawak [18]. Studies through mosquito bites under experimental conditions over the past 10 years in Sarawak by the Malaria Research [23] and there has been one case of a natural infec- Centre, UNIMAS, using nested PCR assays [1, 19], have tion of a woman by P. cynomolgi in Peninsular Malaysia confrmed 890 cases of P. knowlesi and only 6 cases of P. [30]. Both P. cynomolgi and P. inui have been described malariae [20]. Since all 6 patients were Sarawakians who in wild macaques in Sarawak [31]. Since these parasites had been working in logging camps overseas in malaria occur in the same reservoir host as P. knowlesi, there is endemic countries and had recently returned to Sarawak, a strong possibility that natural infections in humans of it indicated that these infections were acquired overseas these simian malaria parasites could occur in Sarawak. and there were no indigenous cases of P. malariae in However, since P. inui is morphologically identical with P. Sarawak. malariae and P. cynomolgi is morphologically similar to Human infections with P. knowlesi cause a wide spec- P. vivax by microscopy [23], these infections have prob- trum of disease, including fatal infections [3, 5, 21]. ably been identifed by routine microscopy as P. malar- Knowlesi malaria induced by mosquito bites under iae or P. vivax. Furthermore, human infections with P. laboratory conditions with the same P. knowlesi strain inui and P. cynomolgi largely resulted in mild infections, produced varying outcomes in humans that included which would not normally require hospitalization [23]. refractory individuals, those that self cured, and others Te recent development of molecular detection methods that required anti-malarial treatment as their infections for simian malaria parasites [31], afords the possibility of were deemed severe [22, 23]. Asymptomatic malaria examining human samples for simian malaria parasites infections have been described for P. falciparum and other than P. knowlesi. Studies utilizing these molecular P. vivax. Although all Plasmodium species can cause detection assays on communities living close to the forest asymptomatic malaria, there have not been many reports fringe, such as those in Betong, would indicate whether of asymptomatic P. malariae [24] and P. ovale [25] infec- other simian malaria parasites are being transmitted to tions. Plasmodium malariae infection may be asympto- humans. Questions relating to whether there are asymp- matic or cause only mild symptoms for many years after tomatic infections, whether there is clustering of cases the initial infection [24]. Detection of these asymptomatic within longhouse communities and whether there is carriers by highly sensitive molecular detection assays human-to-human transmission can be answered by con- has provided a greater understanding of the epidemiol- ducting longitudinal studies in these communities utiliz- ogy of malaria [26, 27]. Studies on P. knowlesi in Sarawak ing molecular detection tools. have been solely hospital-based investigations [1, 5, 8, In this longitudinal study, dried blood spots from 9, 21], but there have been reports of asymptomatic P. individuals living within communities with reported knowlesi infections in Vietnam [28] and more recently malaria cases were repeatedly sampled for the presence in Sabah, Malaysian Borneo [29]. In the Vietnam study, of malaria parasites. Tese longhouse communities were species-specifc SSU rRNA nested PCR screen of 95 in the Betong Division, one of the 12 administrative divi- randomly selected P. malariae samples by PCR revealed sions in Sarawak, in which 161 knowlesi malaria cases 5 to be mixed infections with P. knowlesi. All of them, had been reported in the 3 years preceding the initiation including their family members, were asymptomatic at of this study [18]. sampling time [28]. In the Sabah study, 1147 samples were collected from randomly selected villagers resid- Methods ing at same village as the microscopy-positive patients Study population and blood sample collection as well as members of the patients’ and the selected vil- Participants were recruited from 8 longhouses with resi- lager’s households. P. knowlesi was detected in 20/1147 dents having a history of recent admission with knowlesi (1.7%) samples screened by SSU rRNA nested PCR malaria at Betong hospital (Fig. 1). A review of malaria assays. Te use of cytochrome b nested PCR, SSU rRNA cases in the Betong Division, between 2011 and 2013, real time PCR and chromosome 3 plasmepsin real time identifed the following longhouses with a total of 33 Siner et al. Malar J (2017) 16:417 Page 3 of 11

Fig. 1 Global Positioning System (GPS) coordinates and study identifcation (ID) of the 8 longhouses selected in this study: Nanga Mutok (A, 1˚36′18.1″N, 111˚40′24.7″E, Penebak Ulu (B, 1˚39′9.2″N, 111˚44′15.3″E), Raba Tiput (C, 1˚35′8.2″N, 111˚42′7.5″E), Batu Lintang (D, 1˚30′24.3″N, 111˚36′11.0″E), Begumbang (E, 1˚34′25.4″N, 111˚38′3.4″E), Penyelanih Kiba (F, 1˚33′7.21″N, 111˚37′19.07″E), Nanga Ban (G, 1˚23′46.9″N, 111˚31′4.1″E) and Nanga Keron (H, 1˚27′11.8″N, 111˚37′54.6″E)

knowlesi malaria cases: Nanga Mutok (10 cases), Penebak monkeys while farming, hunting or fshing. Tey were Ulu (2 cases), Raba Tiput (6 cases), Batu Lintang (1 case), also asked whether they felt unwell or feverish on the day Penyelanih Kiba (2 cases), Begumbang (3 cases), Nanga of the blood collection. In addition, temporal artery tem- Ban (1 case) and Nanga Keron (8 cases). All longhouses perature by Termofash­ ® LX-26 (Visiomed, France) was could be accessed by roads, including during the rainy also checked. However, this was only done when it was season. Finger-prick blood samples were taken from each requisitioned in October of 2014 (5th sample collection). person after informed consent/assent had been obtained. Approximately 30 μl of blood was spotted onto Whatman DNA extractions and nested PCR assays 3 M flter papers (GE Healthcare & Life science, U.K.) and Dried blood spots (DBS) collected between January 2014 left to dry at room temperature before being transported and February 2015 (sample collection number 1–7) was to the Malaria Research Centre (MRC) for DNA extrac- extracted based on the pooled strategy of Hsiang [41, tion and molecular detection. Tick blood flms were also 42]. Briefy, four punch holes from two diferent indi- prepared and transported to the MRC for examination viduals were put in one tube and DNA was extracted by by microscopy. Consenting individuals were sampled bi- the Chelex InstaGene™ (Bio-Rad Laboratories, Hercules monthly, from January 2014 until October 2015. Ethical CA, USA) method as described previously [19] in order approval was obtained from the Medical Ethics Commit- to make up DNA samples in Pool 1 (Fig. 2). Next, DNA tee, Faculty of Medicine and Health Sciences, Universiti from two diferent tubes in Pool 1 was combined in a Malaysia Sarawak and the Medical Research and Ethics tube in order to create Pool 2. As shown in Fig. 2, each Committee, Ministry of Health Malaysia. Te Betong tube in Pool 2 now contained 4 samples extracted from 4 Divisional Medical Ofce was informed of all the individ- diferent individuals. uals who were PCR-positive for initiation of anti-malarial Detection for the small subunit ribosomal RNA (SSU treatment. rRNA) gene of Plasmodium was done as previously Study participants were asked of their activities within described [19, 32]. Te frst step of this three-stepped a week prior to the blood collection whether they farmed, pooled screening strategy was to screen using the nested hunted or fshed and also whether they had recently seen PCR assay for the genus Plasmodium. Next, DNA from Siner et al. Malar J (2017) 16:417 Page 4 of 11

Fig. 2 Diagram summarising the pooled strategy used for the screening of DNA extracted from the dried blood spots collected during the frst 5 collections Siner et al. Malar J (2017) 16:417 Page 5 of 11

each sample that made up a genus-positive Pool 2 (e.g.: ­Phusion® (Finnzymes, Espoo, Finland) blunt-end PCR blood spot from individuals A, B, C and D) was re- products using rPLU5 and rPLU6 as the forward and extracted as described previously and tested separately reverse primers. Termocycler (ProFlex™ PCR system for Plasmodium DNA using the nested PCR assay for by Applied Biosystem, USA) parameters were as fol- genus. Lastly, genus positive samples from step two were lows: one cycle of initial denaturation at 98 °C for 30 s; screened by species-specifc nested PCR assays for P. 35 cycles of denaturation (98 °C, 7 s), annealing (63 °C, falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi, P. 20 s) and, extension (72 °C, 17 s); one cycle of fnal exten- cynomolgi and P. inui. As summarized in Fig. 2, pooled sion (72 °C, 10 min). Te 1150 bp amplicon produced was DNA samples were amplifed with the primers rPLU1 visualized on a 1% agarose gel before plasmid ligation and rPLU5 (nest 1 genus assay) and were then screened and cloning using the Zero ­Blunt® TOPO­ ® PCR Clon- using rPLU3 and rPLU4 (nest 2 genus assay). Next, ing Kit (Invitrogen, Carlsbad CA, USA) as per manu- DNA from each of the DBS that made up this Plasmo- facturer’s instructions. Te presence of the correct gene dium positive pool were re-extracted and re-screened by insert was checked by extracting the plasmid using Pure- nested PCR assay for genus. Te nest 1 product from the Link™ Quick Plasmid Miniprep (Invitrogen, Carlsbad genus assay, that was detected as containing Plasmodium CA, USA) followed by an EcoR I (New England Biolabs, DNA, would then be used as the template for the screen USA) digestion and visualization on a 1% agarose gel. with species-specifc primers for P. falciparum, P. vivax, Te plasmid DNA containing the 1150 bp gene insert P. malariae, P. ovale [19], P. knowlesi [1], P. cynomolgi and was sequenced using the ABI Big ­Dye® Terminator Cycle P. inui [31]. Sequencing Kit (Applied Biosystem, USA) with M13F Positive control dried blood spots, made from 30 μl (forward primer) and M13R (reverse primer). Te for- ring-form cultured P. falciparum clone 3D7 parasites ward and reverse primer sequences were removed using diluted with uninfected whole blood to produce parasite SeqMan (DNASTAR, Madison, Wisconsin USA) and densities ranging from 10 to 10,000 parasite per micro- put into NCBI BLAST­ ® in order to identify the sequence litre of blood, were included in the DNA extractions of based on its similarity with the other sequences available blood spots taken from the longhouses. Negative control in the GenBank database. Next, MegAlign (DNASTAR, dried blood spots, made from 30 μl uninfected whole Madison, Wisconsin USA) was used to align the partial blood spotted onto flter papers, were also included in sequences with other Plasmodium sequences in Gen- these extractions. Bank using the Clustal W method. Phylogenetic trees were then constructed using the Neighbour-Joining (NJ) Staining and examination of blood flms method using MEGA 5.05 [34] with bootstrap percentage Tick blood flms from samples positive for malaria infec- based on 1000 replications. All sequences generated were tion by nested PCR assays were stained with 3% Giemsa also submitted to GenBank. for 45 min, air dried, mounted using Eukitt­ ® Quick-hard- ening mounting medium (Sigma-Aldrich, Germany) and Results examined by microscopy to observe for parasites and the Each of the eight longhouses was visited for a total of 10 parasite life cycle stage. Te number of parasites in 1 μl times during the 21-month study period (Table 1). A total of blood was calculated based on the formula below [33]: of 3002 dried blood spots (DBS) and thick blood flms Number of parasites were collected from 555 individuals, in which 289 of the Parasitaemia (p/µl) = recruited participants (52%) and 1697 of the samples col- Number of white blood cells lected (56.52%) were from adults. Although the number × 8000 of study participants started to reduce from the 5th sam- Photos of malaria parasites observed were captured pling trip, 257 DBS were collected in three out of the 6 using a 500 megapixel colour CCD camera (model DP21) remaining sampling trips (Table 1). Females made up 288 and Cell^B software (Olympus, America). of the recruited subjects (52%) and 2642 of the samples collected (88%). Sequencing and phylogenetic analysis of the SSU rRNA One hundred thirty-two of the 267 adult males (49.4%) gene recruited in this study reported participating in farming All samples positive for Plasmodium DNA were selected or hunting activities while the rest spent most their time for cloning and sequencing of the SSU rRNA gene as pre- at school or remained within the vicinity of the long- viously described [1, 4] with slight modifcations. Briefy, house. None of the adult females reported participating the product of the nest 1 genus assay of InstaGene™- in hunting activities. Among the adults who farmed, all extracted DNA was used as the template to generate reported either seeing or hearing monkeys at their farms Siner et al. Malar J (2017) 16:417 Page 6 of 11

Table 1 Total number of longhouses visited, repeat donors, newly recruited donors and blood samples collected for each sampling trip Sampling number 1 2 3 4 5 6 7 8 9 10 11 Total

Number of longhouses visited 5 8 8 8 8 8 8 7 8 8 4 Number of repeat ­donorsa – 194 284 265 310 269 252 255 250 256 112 2447 Number of newly recruited ­donorsb 256 155 42 39 28 19 5 2 4 1 4 555 Total number of blood samples collected­ a, b 256 349 326 304 338 288 257 257 254 257 116 3002 a Total number of repeat donors b Total number of newly recruited subjects or that their crops had been eaten, presumably by these these 7 P. knowlesi positive subjects was 45 years, with monkeys. Tose who hunted reported that they went 5 of them above the age of 60. Of the seven P. knowlesi hunting in highly forested areas. infected subjects, 6 were males. Te P. vivax-infected A pooled DNA strategy was used to screen 2118 of individual was a male, while the Plasmodium positive the 3002 DBS collected between January 2014 and Feb- sample was from a female subject. Except for sample ruary 2015 (sample trip numbers 1–7, Table 2). Eight A008, none of those listed in Table 3 were febrile and of 2118 samples (0.37%) were positive for Plasmodium none of them sought medical treatment at Betong Hos- DNA. Screening with species specifc primers identifed pital. Microscopic examination of the thick blood flms seven P. knowlesi infections and one P. vivax infection. from all 9 subjects revealed malaria parasites in 5 of Since very few cases had been detected using the pooled them: A008, C024, C031, D001 and H011 (Fig. 3). In this DNA strategy, DNA from each of the 884 DBS collected study, no one was infected on more than one occasion, between April and October of 2015 were screened indi- none lived in the same household and none of their fam- vidually (non-pooled DNA screening method) in which ily members were found to be infected (Table 3). one sample was detected as positive for Plasmodium One symptomatic P. knowlesi infection was detected DNA (H033, Table 2). However, the Plasmodium species by nested PCR assays in this study, a 24-year-old male for sample H033 could not be determined by the species- (A008) who sought medical treatment at Betong Hospi- specifc nested PCR assays (Table 3). Te median age of tal the day after his blood sample had been taken. At the

Table 2 Nested PCR assays using Plasmodium genus and species-specifc primers Sample trip Number samples Pool 2­ a Pool 1­ b Genus positive Speciesd number tested samples samples only­ c Pk Pf Pm Po Pv Pin Pcy

1 256 5 3 0 0 0 0 0 0 0 0 2 349 3 3 0 3 0 0 0 0 0 0 3 326 4 4 0 2 0 0 0 1 0 0 4 304 5 1 0 0 0 0 0 0 0 0 5 338 3 5 0 2 0 0 0 0 0 0 6 288 0 0 0 0 0 0 0 0 0 0 7 257 0 0 0 0 0 0 0 0 0 0 8 257 ND ND 1 0 0 0 0 0 0 0 9 254 ND ND 0 0 0 0 0 0 0 0 10 257 ND ND 0 0 0 0 0 0 0 0 11 116 ND ND 0 0 0 0 0 0 0 0 Total 3002 66 86 1 7 0 0 0 1 0 0

ND not done a Pool 2 samples contained DNA extracted from 4 persons b Pool 1 samples contained DNA extracted from 2 persons c The number of individually extracted DNA samples which were positive for Plasmodium genus-specifc primers but negative for all of the Plasmodium species- specifc primers d The number of samples which were positive for Plasmodium species-specifc primers: P. knowlesi: Pk; P. falciparum: Pf; P. malariae: Pm; P. ovale: Po; P. vivax: Pv; P. inui: Pin; P. cynomolgi: Pcy Siner et al. Malar J (2017) 16:417 Page 7 of 11

Table 3 Comparison of nested PCR assay and microscopy results of Plasmodium-infected study participants Study ID Gender Age Occupation Malaria history Temperature Blood collection Microscopy Nested PCR (years) (year of infection) period (parasitaemia)

D001 M 65 Farmer None Afebrile March 2014 32 p/μl Pk H011 M 86 Farmer None Afebrile March 2014 48 p/μl Pk C028 M 45 Farmer None Afebrile March 2014 48 p/μl Pk C024 M 35 Farmer None Afebrile June 2014 Negative Pk C031 M 61 Farmer None Afebrile June 2014 48 p/µl Pk H030 M 61 Retiree None Afebrile June 2014 Negative Pv A008a M 24 Farmer None Febrile (37.4 °C)b October 2014 3536 p/μl Pk A011 F 42 Farmer Pk (2013) Afebrile October 2014 Negative Pk H033 F 71 Farmer None Afebrile April 2015 Negative Genus positive

A, Nanga Mutok; C, Raba Tiput; D, Batu Lintang; H, Nanga Keron; F, female; M, male; p/μl, number of parasites per microlitre of blood; Pk, Plasmodium knowlesi; Pv, Plasmodium vivax a This individual had worked at the farm/went hunting the week prior to sample collection b Body temperature by Thermofash (Visiomed, France) detection of the temporal artery, which was available from October 2014 onwards

Fig. 3 Representative photographs of P. knowlesi life stages observed in Giemsa-stained thick blood flms from 5 persons: a, b from A008E; c from D001B, d from C028C, e from C031C and f from H011B. With the exception of the photograph labeled b (schizont stage), only the ring form of the parasite was observed (photographs labeled a–f)

time of blood collection (between 6 and 11 pm), his tem- before the blood collection. He was PCR negative for poral artery temperature was 37.4 °C, the lowest in the malaria parasite prior to and preceding this infection. temperature range for fever and he did not report feel- One asymptomatic P. vivax infection from a 61-year- ing unwell. Microscopic examination of the thick blood old male (H030) was detected in this study. No parasites flm revealed a few ring forms (Fig. 3a) and one schizont were observed in his thick blood flm and he did not (Fig. 3b); with an estimated parasitaemia of 3536 para- report feeling unwell at the time of the blood collection. sites/μl of blood. On follow-up, he reported that he had Samples taken prior to and preceding this infection was been clearing land for farming in a forested area the week PCR negative for malaria parasites. Siner et al. Malar J (2017) 16:417 Page 8 of 11

Parasites infecting A008 and C031 were confrmed parasites by microscopy. In comparison, PCR detected as P. knowlesi through phylogenetic analysis of the SSU parasite DNA in samples from 1169 individuals (31.3%) rRNA genes (Fig. 4). However, attempts at sequencing [15]. In another study in Sumatera, 6 out of 1495 asymp- the SSU rRNA gene from samples A011 and C028 were tomatic individuals screened were detected as having unsuccessful. P. knowlesi (n = 1) and P. vivax (n = 3) infections [35]. Screening of 638 (in northeast Myanmar) and 1070 (in Discussion western Tailand) villagers from these hypo-endemic Te fnding of the present study, that sub-microscopic areas showed that microscopy detected 1.3 and 0.04% infections were observed in communities that are hypo- infections compared to nested PCR detecting 1.9 and endemic for malaria, is consistent with reports by oth- 6.2% as Plasmodium positive [36]. A screen of 11,185 ers. In a recent study in Sumatera, Indonesia, 614 of samples in western Kenya for P. falciparum infections 3731 participants (16.5%) were positive for malaria revealed that more false negatives were observed when

S-type SSU RNA

A-type SSU RNA

Fig. 4 Neighbour-joining tree constructed using partial sequences of SSU rRNA genes of Plasmodium species. The sequences generated in the cur‑ rent study are boxed. Bootstrap percentage was based on 1000 replicates and only those above 70% are shown Siner et al. Malar J (2017) 16:417 Page 9 of 11

samples were collected from low malaria transmission the Betong division [18]. More infections could have also as 8% more samples were detected as positive by nested been detected had the sampling intervals been shorter. A PCR assays compared to microscopy [37]. An average 2-month interval between samplings could increase the parasite prevalence of 28.4% (in adults) and 25.5% (in risk of missing transient asymptomatic infections that children) were reported when 9260 microscopy nega- might have resolved spontaneously. Terefore, sampling tive samples from afebrile individuals in Uganda was fewer sites but more frequently might help detecting screened for P. falciparum infections by loop-mediated transient asymptomatic infections within the afected isothermal amplifcation [38]. More recently, a malaria communities. survey of 845 afebrile schoolchildren in northwest Ethio- In contrast to the Sabah study that detected P. knowlesi pia reported 0.95% detection by microscopy compared infections in 15–45 year olds [29] and in young children to 12.7% that were detected as Plasmodium positive by in the Vietnam study [28]; only adults were infected with real time PCR [39]. Only one of the 1147 (0.08%) afebrile malaria in our study. With the exception of one of our individuals from northeast Sabah of Malaysian Borneo PCR-positive study participants, all had farms located that were screened for P. knowlesi was detected as posi- near forested areas. In addition, all reported seeing tive by microscopy compared to 20 individuals (1.7%) by long-tailed macaques or signs of their presence at their nested PCR [29]. Similarly, PCR-based methods detected farms. Te location of the farms and presence of long- more malaria infections in two studies done in diferent tailed macaques is in concordance with “forest exposure” provinces in Sumatera: 20/1532 (0.06%) PCR-confrmed as one of the risks for P. knowlesi infection [3, 8]. Stud- P. knowlesi infections compared to none by microscopy ies in Sabah [29] and Vietnam [28] found asymptomatic [35]; 612/3731 (16.4%) Plasmodium positive by micros- infections to be common in communities afected by P. copy compared to 377/3731 (10%) P. knowlesi positive knowlesi infections due to the co-existence with the vec- by hemi-nested PCR [15]. In the current study, the P. tor and host [8, 28]. Interestingly, two of the knowlesi knowlesi infections detected were either submicroscopic positive individuals (D001 and H011) reported at fol- (n = 2) or had parasitaemia of less than 50 parasites/μl of low-up that they mainly stayed within the longhouse blood (n = 4). compound and that they did not participate in farming, A higher detection of submicroscopic infections could hunting or fshing activities. Both of their longhouses have been achieved had more sensitive methods or a were surrounded by pockets of substantially forested larger amount of blood and subsequent DNA extraction areas. Although this does not preclude undisclosed activ- using commercial kits was undertaken in the current ities that may bring them near forested areas, a yet-to- study. One more sample was detected as positive when be-identifed P. knowlesi vector that has adapted to dwell DNA was prepared from 1708 samples from asympto- within the longhouse compound is a possibility [43]. matic communities using a commercial DNA extraction Te DBS from sample H033 could only be determined kit compared to the standard DNA extraction method as Plasmodium-infected as the species-specifc nested [36]. Detection of more P. vivax infected samples by PCR assays failed to produce an amplicon. Tis could nested reverse transcription PCR (182/1005) or nested be due to the primer design that was based on either PCR (24/1005) was attributed to the amount of template the asexually transcribed (A) or the sexually transcribed used in the assay which was DNA or RNA extracted from (S) forms of Plasmodium SSU rRNA gene for the spe- whole blood samples [40]. Capture and ligation probe- cies-specifc nested PCR assays [44]. Te genus-specifc PCR that used templates extracted from DBS was able to primer pair anneals to both the asexual and sexual forms detect only 19/1005 samples screened [40]. In this study, of the SSU rRNA gene and therefore, nested PCR assays initial DBS screens were done using a high-throughput with the genus-specifc primers are more sensitive than method in order to reduce the turnaround time for infec- those with the species-specifc assays. tion detection [41, 42]. Te conventional method for Te only asymptomatic P. vivax infection was detected screening DBS, in which each DNA sample was assayed in a retired ofshore worker who divided his time between individually, was applied in the later part of our study in his home at the longhouse in Betong and a relative’s an order to improve detection of malaria parasites. Com- home in , a city in the northern region of Sarawak. pared to a detection limit of 100 parasites per microlitre Although P. knowlesi infection is more common in Sabah (p/μl) of blood for the pooled method [41], detection and Sarawak, 7% of microscopy-confrmed malaria cases limit of SSU rRNA nested PCR of individual samples was reported in 2015 were due to P. vivax [18]. 6 p/μl of blood [19]. However, despite using the more Absence of P. cynomolgi and P. inui infection in asymp- sensitive assay later in our study, only one of the 884 DBS tomatic individuals in this study supports the rarity in screened was Plasmodium-positive that could be related reports of individuals hospitalized with these infec- to the decrease in number of reported malaria cases in tions. Only one case of a human naturally infected with Siner et al. Malar J (2017) 16:417 Page 10 of 11

P. cynomolgi has been reported, which was in Peninsu- Funding This work was funded through a research grant from the Malaysia Ministry of lar Malaysia [30], while no natural human infection by P. Education to AS, MZ & BS [Grant Number FRGS/SKK01(01)/1056/2013(02)] and inui has been reported. Human infections by P. cynomolgi by a MyBrain15 scholarship from the Malaysia Ministry of Higher Education to could be limited by its requirement for select receptors LST. on human RBCs [45]. Lack of suitable vectors could also afect invasion of human RBCs by these species. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ Tere may be people with asymptomatic and sub-micro- lished maps and institutional afliations. scopic malaria who may be a source of human-to-human transmission. Asymptomatic malaria could be a problem Received: 4 June 2017 Accepted: 11 October 2017 for malaria elimination since most asymptomatic individu- als remain untreated and do not seek treatment since they do not develop any signs and symptoms of malaria [46]. Current molecular, epidemiological and entomological References evidence suggests that P. knowlesi is a zoonosis in Sarawak 1. Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, et al. A large focus of naturally acquired Plasmodium knowlesi infections but human-to-human malaria cannot be ruled out com- in human beings. Lancet. 2004;363:1017–24. pletely. It may be occurring but proving it is going to be 2. Sinton JA, Mulligan HW. Plasmodium knowlesi. In: The primate malarias. difcult because knowlesi malaria transmission is occur- Bethesda: U.S. National Institute of Allergy and Infectious Diseases [for sale by the Supt. of Docs., U.S. Govt Print Of., Washington]. 1971. p. 317. ring in areas where macaques are found. 3. Singh B, Daneshvar C. Human infections and detection of Plasmodium knowlesi. Clin Microbiol Rev. 2013;26:165–84. Conclusions 4. Lee KS, Cox-Singh J, Singh B. Morphological features and diferential counts of Plasmodium knowlesi parasites in naturally acquired human Tis study showed that asymptomatic P. knowlesi and P. infections. Malar J. 2009;8:73. vivax infections are present in communities with reported 5. Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, et al. cases of knowlesi malaria. Tese infections were either Plasmodium knowlesi malaria in humans is widely distributed and poten‑ tially life threatening. Clin Infect Dis. 2008;46:165–71. submicroscopic or had very low parasitaemia. None of the 6. Vythilingam I, Noorazian YM, Huat TC, Jiram AI, Yusri YM, Azahari AH, et al. asymptomatic P. vivax and P. knowlesi-infected persons Plasmodium knowlesi in humans, macaques and mosquitoes in peninsu‑ sought medical attention and all were afebrile. lar Malaysia. Parasit Vectors. 2008;1:26. 7. Lau YL, Tan LH, Chin LC, Fong MY, Noraishah MA, Rohela M. Plasmodium knowlesi reinfection in human. Emerg Infect Dis. 2011;17:1314–5. Abbreviations 8. Barber BE, William T, Dhararaj P, Anderios F, Grigg MJ, Yeo TW, et al. Epide‑ DBS: dried blood spot; PCR: polymerase chain reaction; SSU rRNA: small miology of Plasmodium knowlesi malaria in north-east Sabah, Malaysia: subunit ribosomal RNA. family clusters and wide age distribution. Malar J. 2012;11:401. 9. William T, Menon J, Rajahram G, Chan L, Ma G, Donaldson S, et al. Severe Authors’ contributions Plasmodium knowlesi malaria in a tertiary care hospital, Sabah, Malaysia. AS and BS designed the study. AS, LST, BS, MZ and FKT conducted the feld Emerg Infect Dis. 2011;17:1248–55. work. LST, KAK, DSAM and FKT conducted the laboratory experiments. AS, 10. Jongwutiwes S, Putaporntip C, Iwasaki T, Sata T, Kanbara H. Naturally BS and LST wrote the manuscript. All authors read and approved the fnal acquired Plasmodium knowlesi malaria in human, Thailand. Emerg Infect manuscript. Dis. 2004;10:2211–3. 11. Putaporntip C, Hongsrimuang T, Seethamchai S, Kobasa T, Limkittikul K, Cui L, et al. Diferential prevalence of Plasmodium infections and Acknowledgements cryptic Plasmodium knowlesi malaria in humans in Thailand. J Infect Dis. The authors would like to thank the Director of Health Malaysia for permission 2009;199:1143–50. to publish this paper. We also thank Dr. Johnny Pangkas, Mr. Joseph Tau Katip, 12. Luchavez J, Espino F, Curameng P, Espina R, Bell D, Chiodini P, et al. Human Mr. Rahynold Rehengia Gagat, Mr. Joshua Edward Egam and Mr. Ezra Tan Dao infections with Plasmodium knowlesi, the Philippines. Emerg Infect Dis. Quan for assistance in the feld; the respective longhouse communities in the 2008;14:811–3. Betong Division for their support and contribution to this project. 13. Ng OT, Ooi EE, Lee CC, Lee PJ, Ng LC, Pei SW, et al. Naturally acquired human Plasmodium knowlesi infection, Singapore. Emerg Infect Dis. Competing interests 2008;14:814–6. The authors declare that they have no competing interests. 14. Jiang N, Chang Q, Sun X, Lu H, Yin J, Zhang Z, et al. Co-infections with Plasmodium knowlesi and other malaria parasites, Myanmar. Emerg Infect Availability of data and materials Dis. 2010;16:1476–8. The DNA sequences generated in this study are deposited in GenBank with 15. Lubis IN, Wijaya H, Lubis M, Lubis CP, Divis PC, Beshir KB, et al. Contribu‑ Accession Numbers: KY404056, KY404057, KY404058 and KY404059. tion of Plasmodium knowlesi to multispecies human malaria infections in North Sumatera, Indonesia. J Infect Dis. 2017;215:1148–55. Consent for publication 16. Khim N, Siv S, Kim S, Mueller T, Fleischmann E, Singh B, et al. Plasmodium All authors have seen and approved the manuscript and its contents, and knowlesi infection in humans, Cambodia, 2007–2010. Emerg Infect Dis. have agreed to submission for publication. 2011;17:1900–2. 17. Marchand RP, Culleton R, Maeno Y, Quang NT, Nakazawa S. Co-infections Ethics approval and consent to participate of Plasmodium knowlesi, P. falciparum, and P. vivax among humans Ethical approval for this work was obtained from the Medical Ethics Com‑ and dirus mosquitoes, Southern Vietnam. Emerg Infect Dis. mittee, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak 2011;17:1232–9. (UNIMAS/TNC(AA)-03.02/06-11/1 (41)) and the Medical Research and Ethics 18. Ofcial Portal: Sarawak State Health Department. http://jknssarawak.moh. Committee, Ministry of Health Malaysia (NMRR-10-1194-7854). gov.my/bm/. Siner et al. Malar J (2017) 16:417 Page 11 of 11

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